Genetic Data Show That Carcharhinus Tilstoni Is Not Confined to the Tropics

Journal of Fish Biology (2010) 77, 1165–1172 doi:10.1111/j.1095-8649.2010.02770.x, available online at wileyonlinelibrary.com

Genetic data show that Carcharhinus tilstoni is not conﬁned to the tropics, highlighting the importance of a multifaceted approach to species identiﬁcation

J. J. Boomer*†, V. Peddemors‡ andA.J.Stow*

*Department of Biological Sciences, Macquarie University, Sydney, NSW 2109, Australia and ‡Cronulla Fisheries Research Centre of Excellence, P.O. Box 21, Cronulla, NSW 2230, Australia

(Received 26 June 2010, Accepted 9 August 2010)

This study shows a range extension for the Australian blacktip shark Carcharhinus tilstoni,which was believed to be restricted to Australia’s tropical waters, of >1000 km into temperate waters, revealing its vulnerability to a wider commercial ﬁshery. © 2010 The Authors Journal compilation © 2010 The Fisheries Society of the British Isles

Key words: blacktip shark; Carcharhinus limbatus; mtDNA; shark ﬁshery; species distribution.

Sharks are proving to be especially vulnerable to anthropogenic pressures, in part this is a response to the K-selected natural-history traits that characterize many species along with pressure from targeted and non-targeted fishing activities (Stevens et al., 2000). In many cases, species distributions span state and international management boundaries, across which protection and management processes may vary for any given species (Last & Stevens, 2009). Therefore, effective conservation and manage ment require knowledge of species distributions. Despite the size and notoriety of sharks, distributions of some species remain uncertain due to limited opportunities for observation or difficulties with species identification. Sharks, especially those in the carcharhinid family, can be very difficult to identify to species level using morphological features (Chan et al., 2003). Distinguishing features for some species are few, and sharks caught by commercial fishers often have important features removed (e.g. head and teeth) before being landed ashore, making reliable identification very difficult (Chan et al., 2003; Last & Stevens, 2009). The fin trade exacerbates problems with identifying harvested sharks. A solution has been found with the development of DNA markers for species identification. Over the last decade, rapid improvements in DNA technology have seen a suite of markers developed which require only a small sample of tissue for reliable species identification (Chapman et al., 2003; Greig et al., 2005; Ward et al., 2008). The use of this technology has resulted in further taxonomic revision and improved

†Author to whom correspondence should be addressed. Tel.: +61 2 9850 8143; fax: +61 2 9850 7972; email: [email protected] 1165 © 2010 The Authors Journal compilation © 2010 The Fisheries Society of the British Isles 1166 J. J. BOOMER ET AL . knowledge of shark species distributions (Gardner & Ward, 2002; Quattro et al., 2006; Corrigan et al., 2008). There are two species of shark referred to by the common name blacktip shark, the common blacktip shark Carcharhinus limbatus (Muller¨ & Henle) and the Australian blacktip shark Carcharhinus tilstoni (Whitley). These species are distinguished by vertebral counts and forensic methods (Lavery & Shaklee, 1991; Last & Stevens, 2009). Carcharhinus limbatus has a global distribution while C. tilstoni is believed to be restricted to the tropical waters off northern Australia (Fig. 1) where it occurs in sympatry with C. limbatus (Last & Stevens, 2009). Blacktip sharks are taken in commercial fisheries worldwide. It is generally accepted that C. limbatus is taken in most regions while in northern Australia both C. limbatus and C. tilstoni are taken by commercial fisheries (Ovenden et al., 2010). At present, no external morphological features are available to distinguish these species and therefore poor knowledge of catch rates compromises the development of sustainable fisheries. DNA technologies provide a solution to this problem (Shivji et al., 2002). Blacktip sharks are within the top five shark species commercially harvested in temperate waters of New South Wales (NSW) on the Australian east coast (Macbeth et al., 2009). Additionally, they are caught in this region by the shark beach meshing programme, which operates between September and May off 51 popular beaches between Newcastle and Wollongong (Green et al., 2009a). Historically, blacktip sharks occurring in NSW waters have been classified as C. limbatus (Green et al., 2009a). The species identity of blacktip sharks occurring in NSW, however, has

Sydney

N km 0 312·5 625 1250 1875 2500

Fig. 1. Currently recognized distribution of Carcharhinus tilstoni ( ), which is known from Thevenard Island (Western Australia) to Rockhampton (Queensland) along the continental shelf of tropical Australia (Last & Stevens, 2009). ( ), area from which samples for this study were collected.

© 2010 The Authors Journal compilation © 2010 The Fisheries Society of the British Isles, Journal of Fish Biology 2010, 77, 1165–1172 RANGE EXTENSION OF CARCHARHINUS TILSTONI 1167 never been thoroughly investigated. In this study, genetic approaches were used to identify species of blacktip sharks occurring off Sydney, NSW, Australia. Tissue samples were collected from sharks caught in the NSW Shark Meshing (Bather Protection) Program (NSWDPI, 2009) (Fig. 1). DNA was extracted using a modified salting out technique (Sunnucks & Hales, 1996). Two mitochondrial genes (COI and ND4 ) were amplified. The cytochrome oxidase I (COI ) gene was ampli fied with the primers FishF1Mod 5� ACC AAC CAC AAA GAY ATY GGC AC 3� (modified from Ward et al., 2005) and FishR15� TAG ACT TCT GGG TGG CCA AAG AAT CA 3� (Ward et al., 2005). The sodium dehydrogenase subunit 4 (ND4 ) gene was amplified with primers designed in this study MaND4F 5� ACC MAA AGC YCA CGT WGA AGC 3� and MaND4R 5� TCT TGC TTG GAG TTG CAC CA 3�. These genes were selected for analysis because they have previously been successfully applied to distinguish C. limbatus and C. tilstoni (Ovenden et al., 2010). The COI gene was used to initially screen samples. A total of 54 samples identified by field observers as some form of carcharhinid shark were screened, 13 of these were identified as C. limbatus and five as C. tilstoni based on COI.Asa prior study suggested that C. limbatus and C. tilstoni may share haplotypes at the COI gene (Wong et al., 2009), these 18 samples were further investigated using the ND4 gene to confirm their species identity. For both genes, PCR reactions were in 15 μL volumes and contained ×1 PCR buffer, 2 mM MgCl2,0·2 mM of each deoxynucleoside triphosphate (dNTP), 0·5 μM of each primer, 1 U Taq DNA poly merase (Promega; www.promega.com) and 1 μL DNA template. Cycling conditions ◦ consisted of an initial denaturation step at 94 C for 3 min, followed by a touch ◦ down PCR with six cycles, decreasing the annealing temperature by 1 C per cycle. ◦ ◦ Denaturation was at 94 C (30 s), annealing at 60 to 55 C (1 min) and extension ◦ ◦ ◦ ◦ at 72 C (1 min). A cycle of 94 C(30s),55 C(30s) and72 C(1min)was ◦ then repeated 30 times, followed by a final extension of 72 C for 5 min. The PCR product was purified with EXOSAP-IT (USB; www.usbweb.com) and sequenced in an ABI 377 automated DNA sequencer. DNA sequences were assembled and aligned in MEGA version 4 (Tamura et al., 2007) with reference sequences for C. tilstoni and C. limbatus as well as reference sequences for the graceful shark Carcharhinus amblyrhynchoides (Whitley) and the bull shark Carcharhinus leucas (Muller¨ & Henle). Carcharhinus amblyrhynchoides has been shown to be very closely related to C. limbatus and was included in analyses to ensure this species was not present in the sample set (Ward et al., 2008; Ovenden et al., 2010). Reference sequences for each of these species were obtained for the COI region from GenBank (GenBank accession numbers: C. tilstoni, DQ108283; C. limbatus, EU39862; C. amblyrhynchoides, EF609307 and C. leucas, EF609311). The reference sequences for the ND4 gene for each species except C. leucas were obtained from Ovenden et al. (2010) (GenBank accession numbers: C. limbatus, GQ227272; C. tilstoni, GQ227268; C. amblyrhynchoides, GQ227276). The ND4 ref erence sequence for C. leucas was obtained as part of this study. The ND4 and COI sequences were concatenated for phylogenetic analysis giving a final sequence of 1353 base pairs (bp). Phylogenetic relationships were inferred using the neighbour joining (NJ) distance method implemented in MEGA 4 (Tamura et al., 2007) and the Bayesian method implemented in MrBayes 3.1 (Ronquist & Huelsebeck, 2003). For the NJ approach, the best model of nucleotide substitution was determined to be a Tamura–Nei model (TrN) using Akaike information criterion with the software

FindModel (LANL, 2009), which is an internet-based application of the programme ModelTest (Posada & Crandall, 1998). Reliability of tree nodes for the NJ tree was assessed using 10 000 bootstrap replicates. The Bayesian approach to tree building was completed in MrBayes 3.1 (Ronquist & Huelsebeck, 2003) using a GTR + G substitution model. Four chains were run for 500 000 generations and a consensus tree was constructed. Carcharhinus leucas was used as the out-group for each of these phylogenetic analyses. Commercially harvested sharks, particularly species with slow growth and low fecundity such as the carcharhinids, are in dire need of well-developed management plans to slow population declines (Field et al., 2009). Of fundamental importance is the species identification. This study highlights the benefits of applying genetic approaches to species identification. The results show that the range of C. tilstoni extends into temperate waters, >1000 km further south than previously known, and that at least two species of blacktip sharks (C. limbatus and C. tilstoni ) occur in the temperate waters off Sydney, Australia. The topology of the phylogenetic trees clearly supports the presence of these two species using both the NJ and Bayesian approaches. Two clades of NSW blacktip sharks belonging to each of the two species were supported by high bootstrap values in the NJ tree (Fig. 2) and high probabilities

NSW 25 NSW 98 NSW 105 NSW 17 NSW 177 NSW 64 NSW 228 NSW 276 NSW 291 NSW 244 NSW 188 99 NSW 18 NSW 336 Carcharias limbatus Carcharias amblyrhynchoides NSW 23 NSW 154 Carcharias tilstoni

99 NSW 191 NSW 126 86 NSW 132 Carcharias leucas

0·01

Fig. 2. Neighbour-joining tree of mitichondrial (mt)DNA sequences. Reference sequences are designated by species name. Bootstrap values are given where values are >80.

© 2010 The Authors Journal compilation © 2010 The Fisheries Society of the British Isles, Journal of Fish Biology 2010, 77, 1165–1172 RANGE EXTENSION OF CARCHARHINUS TILSTONI 1169 of clade credibility in the Bayesian tree (>95%). One clade consisting of 13 indi viduals grouped with the reference sequence for C. limbatus and the other with five individuals grouped with the reference sequence for C. tilstoni. No samples from NSW were found to group with the reference sequence from C. amblyrhynchoides. Three COI haplotypes were found, two of which were associated with C. limbatus and one with C. tilstoni.FiveND4 haplotypes were found, two of which were associ ated with C. limbatus and three with C. tilstoni. All mutations were single nucleotide substitutions and predominantly transitions (Tables I and II). Between C. limbatus and C. tilstoni, 13 fixed differences at ND4 and three fixed differences at COI were found. Ten of the ND4 characters and two of the COI characters correspond to the fixed differences identified by Ovenden et al. (2010), therefore supporting the use of these characters as diagnostic features. The remaining differences were not fixed between species (Ovenden et al., 2010). The overall level of sequence divergence between C. tilstoni and C. limbatus fur ther supports species identification. For the 1353 bp of sequence data, an average sequence divergence of 1·5% occurred between NSW samples of C. tilstoni and C. limbatus. Average sequence divergence between NSW C. tilstoni and the refer ence sequence for C. tilstoni was low (0·2%) as was that between NSW C. limbatus and the C. limbatus reference sequence (0·2%). Sampling as a part of the NSW shark meshing programme has predominantly been opportunistic to date and as such accurate predictions about the relative abundance of these two species in NSW waters are unable to be made. Proportions of each species collected in this study, however, do suggest that C. limbatus may be more abundant of the two near Sydney. Because C. limbatus and C. tilstoni differ in aspects of their growth and reproduction (Last & Stevens, 2009), they may require different management strategies. As both of these species probably comprise part of the catch in commercial fisheries in NSW waters, a more detailed study of the relative composition of these species is urgently required to evaluate the sustainability of current shark fishing practices. This work illustrates how difficulties involved with identifying carcharhinids on the basis of morphology can be resolved with a genetic approach. The finding of an additional species of carcharhinid in temperate waters off Australia suggests

Table I. Nucleotide substitutions at the cytochrome oxidase I (COI ) gene. The position of each of these diagnostic substitutions has been adjusted so that the base pair count corresponds to those presented by Ovenden et al. (2010). GenBank accession numbers are given for reference haplotypes and haplotypes new to this study. New haplotypes identiﬁed in this study are indicated in bold

Accession COI number 256 292 394 520 n Carcharhinus limbatus COI 1 C C CG 12 C. limbatus COI 2 HM231107 T 1 C. limbatus GenBank EU398625 Carcharhinus tilstoni COI 1 T T A 5 C. tilstoni GenBank DQ108283 T T A n, the number of individuals with each haplotype.

1 n G haplotypes are designated

limbatus A G Carcharhinus T al . (2010). et

) gene. The position of each of these diagnostic substitutions has ND4 T . Reference sequences used for construction of phylogenetic trees are designated

til C.

haplotypes are designated as TCC TC TCT CCCCCTAGCATCC12 CCCCCTAGCATCC12 TCT TC TCC CC T T T T C C T T C C T T C T T T T T T C C C C A A G G C C T T T T 1 1 tilstoni . GenBank accession numbers are given for each haplotype. New haplotypes identiﬁed in this study are indicated in bold

ref

til C. number 76 85 115 211 217 232 259 283 293 298 308 325 358 367 388 463 523 531 550 671 775 Accession GQ227272 GQ227273 GQ227271 TGQ227268 T C T C T C C T C T T T T T T C T C C A C T A G C T G C T T 3 T HM231105 HM231106 HM231104 and Carcharhinus

ref II. Nucleotide substitutions at the sodium dehydrogenase subunit 4 ( 1 and 2 ref 2 3 1 ref

lim til lim til til til lim lim lim able , the number of individuals with each haplotype. C. C. ND4 C. C. C. C. C. C. T C. n been adjusted so that the base pair count corresponds to those presented by Ovenden

© 2010 The Authors Journal compilation © 2010 The Fisheries Society of the British Isles, Journal of Fish Biology 2010, 77, 1165–1172 RANGE EXTENSION OF CARCHARHINUS TILSTONI 1171 taxonomic uncertainty with carcharhinid catches worldwide. Furthermore, this study demonstrates how scientific benefits can be gained from shark meshing programmes and fisheries by the low cost practice of collecting and preserving small amounts of tissue.

We would like to thank D. Reid and the NSW shark meshing observers for the collection of tissue samples, S. Dixon for assistance in the laboratory, J. Ovenden, J. Morgan and I. Field for providing sequence data and valuable advice and K. Rowling for useful comments on the manuscript. This work was funded by NSW Industry and Investment (formerly NSW DPI).

References Chan, R. W. K., Dixon, P. I., Pepperell, J. G. & Reid, D. D. (2003). Application of DNA- based techniques for the identification of whaler sharks (Carcharhinus spp.) caught in protective beach meshing and by recreational fisheries off the coast of New South Wales. Fishery Bulletin 101, 910–914. Chapman, D. D., Abercrombie, D. L., Douady, C. J., Pikitch, E. K., Stanhope, M. J. & Shivji, M. S. (2003). A streamlined, bi-organelle, multiplex PCR approach to species identification: application to global conservation and trade monitoring of the great white shark, Carcharodon carcharias. Conservation Genetics 4, 415–425. Corrigan, S., Huveneers, C., Schwartz, T. S., Harcourt, R. G. & Beheregaray, L. B. (2008). Genetic and reproductive evidence for two species of ornate wobbegong shark Orec tolobus spp. on the Australian east coast. Journal of Fish Biology 73, 1662–1675. Field, I. C., Meekan, M. G., Buckworth, R. C., Bradshaw, C. J. A. & David, W. S. (2009). Susceptibility of sharks, rays and chimaeras to global extinction. Advances in Marine Biology 56, 275–363. Gardner, M. G. & Ward, R. D. (2002). Taxonomic affinities within Australian and New Zealand Mustelus sharks (Chondrichthyes: Triakidae) inferred from allozymes, mito chondrial DNA and precaudal vertebrae counts. Copeia 2002, 356–363. Green, M., Ganassin, C. & Reid, D. D. (2009). Report into the NSW Shark Meshing (Bather Protection) Program, Incorporating a Review of the Existing Program and Environmen tal Assessment. Cronulla: NSWDPI Fisheries Conservation and Aquaculture Branch. Greig, T. W., Moore, M. K. & Woodley, C. M. (2005). Mitochondrial gene sequences useful for species identification of western North Atlantic Ocean sharks. Fishery Bulletin 103, 516–523. Last, P. R. & Stevens, J. D. (2009). Sharks and Rays of Australia, 2nd edn. Melbourne: CSIRO Publishing. Lavery, S. & Shaklee, J. B. (1991). Genetic evidence for separation of two sharks Carcharhi nus limbatus and C. tilstoni from northern Australia. Marine Biology 108, 1–4. Macbeth, W. G., Geraghty, P. T., Peddemors, V. M. & Gray, C. A. (2009). Observer-based study of targeted commercial fishing for large shark species in waters off northern New South Wales, Final Report to the Northern Rivers Catchment Management Author ity. Industry & Investment NSW Fisheries Final Report Series. Cronulla: Industry & Investment NSW. Ovenden, J., Morgan, J., Kashiwagi, T., Broderick, D. & Salini, J. (2010). Towards better management of Australia’s shark fishery: genetic analyses reveal unexpected ratios of cryptic blacktip species Carcharhinus tilstoni and C. limbatus. Marine and Freshwater Research 61, 253–262. Posada, D. & Crandall, K. A. (1998). ModelTest: testing the model of DNA substitution. Bioinformatics 14, 817–818. Quattro, J. M., Stoner, D. S., Driggers, W. B., Anderson, C. A., Priede, K. A., Hoppmann, E. C., Campbell, N. H., Duncan, K. M. & Grady, J. M. (2006). Genetic evidence of cryptic speciation within hammerhead sharks (genus Sphyrna). Marine Biology 148, 1143–1155. Ronquist, F. & Huelsebeck, J. (2003). MrBayes 3: Bayesian phylogenetic inference under mixed models. Bioinformatics 19, 1572–1574.

Shivji, M., Clarke, S., Pank, M., Natanson, L., Kohler, N. & Stanhope, M. (2002). Genetic identification of pelagic shark body parts for conservation and trade monitoring. Con servation Biology 16, 1036–1047. Stevens, J. D., Bonfil, R., Dulvy, N. K. & Walker, P. A. (2000). The effects of fishing on sharks, rays, and chimaeras (chondrichthyans), and the implications for marine ecosys tems. ICES Journal of Marine Science 57, 476–494. Sunnucks, P. & Hales, D. F. (1996). Numerous transposed sequences of mitochondrial cyto chrome oxidase I-II in aphids of the genus Sitobion (Hemiptera: Aphididae). Molecular Biology and Evolution 13, 510–524. Tamura, K., Dudley, J., Nei, M. & Kumar, S. (2007). MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. Molecular Biology and Evolution 24, 1596–1599. Ward, R. D., Zemlak, T. S., Innes, B. H., Last, P. R. & Hebert, P. D. N. (2005). DNA barcoding Australia’s fish species. Philosophical Transactions of the Royal Society B 360, 1847–1857. Ward, R. D., Holmes, B. H., White, W. T. & Last, P. R. (2008). DNA barcoding in Aus tralasian chondrichthyans: results and potential uses in conservation. Marine and Fresh water Research 59, 57–71. Wong, E. H. K., Shivji, M. S. & Hanner, R. H. (2009). Identifying sharks with DNA bar codes: assessing the utility of a nucleotide diagnostic approach. Molecular Ecology Resources 9, 243–256.

Electronic References LANL (2009). HIV Sequence Database: FindModel. Los Alamos, NM: Los Alamos National Laboratory. Available at http://www.hiv.lanl.gov/content/sequence/findmodel/find model.html/. Accessed May 2010. NSWDPI (2009). NSW shark meshing (bather protection) program. Available at http://www. dpi.nsw.gov.au/ data/assets/pdf file/0014/101327/NSW-shark-meshing-program.pdf. Accessed May 2010.